Project Summary The methods of synthetic biology have transformed practice throughout the biological sciences, but have yet to find wide application in neurobiology. This is in part because many signaling receptors and pathways in the brains are shared, limiting the latitude for narrowly targeted engineering strategies. To create a wider range of tools for selective cell modulation, we propose to develop directed evolution methods that will generate orthogonal neural receptors that respond to cannabinoids and offer multiple different chemogenetic control points across the brain and thereby open the way to a synthetic neurobiology. The proposed methods should yield very High Affinity receptors, that have Validated Orthogonalities for their receptor:ligand Couples. Our HAVOCs will stand in contrast to current DREADD and DART approaches in that they will allow the use of natural effectors, but at much lower concentrations, in essence flying below the ?radar cover? of endogenous receptors in the brain. In particular, we will use HAVOCs to examine the gate theory of pain, and in consequence serve as a surrogate model for targeted nano-dosing strategies for cannabinoids to safely promote analgesia and combat addiction. As a starting point for the development of nano-dosing strategies, we will focus on the CB2 receptor, which is sparsely expressed in the brain, but which has known functions in inhibiting dopaminergic neurons. Using our novel directed evolution method, Compartmentalized Partnered Replication (CPR), we will initially evolve individual variants of CB2 that can interact with high affinity with the cannabinoids b-caryophyllene, cannabidiol (CBD), and other minor cannabinoids (Aim 1). We will proof the utility of these compounds and their evolved receptors with isolated neurons and directly in a mouse model for pain (Aim 2). While movement to the clinic will ultimately require introduction of novel receptors into patients, likely via gene therapies, the ability to target protein production in particular neural pathways may provide one of the few viable methods for the chronic treatment of pain. Into the future, the directed evolution strategies we have developed are fungible between multiple different receptors and receptor types, and we suggest that HAVOCs may therefore serve as generalizable neurotechnological tools to understand and manipulate a variety of neural functions.